Treatment Of Immune-mediated Diseases Research Articles

Impact of platelet lysate on immunoregulatory characteristics of equine mesenchymal stromal cells.

Multipotent mesenchymal stromal cells (MSC) play an increasing role in the treatment of immune-mediated diseases and inflammatory processes. They regulate immune cells via cell-cell contacts and by secreting various anti-inflammatory molecules but are in turn influenced by many factors such as cytokines. For MSC culture, platelet lysate (PL), which contains a variety of cytokines, is a promising alternative to fetal bovine serum (FBS). We aimed to analyze if PL with its cytokines improves MSC immunoregulatory characteristics, with the perspective that PL could be useful for priming the MSC prior to therapeutic application. MSC, activated peripheral blood mononuclear cells (PBMC) and indirect co-cultures of both were cultivated in media supplemented with either PL, FBS, FBS+INF-γ or FBS+IL-10. After incubation, cytokine concentrations were measured in supernatants and control media. MSC were analyzed regarding their expression of immunoregulatory genes and PBMC regarding their proliferation and percentage of FoxP3+ cells. Cytokines, particularly IFN-γ and IL-10, remained at high levels in PL control medium without cells but decreased in cytokine-supplemented control FBS media without cells during incubation. PBMC released IFN-γ and IL-10 in various culture conditions. MSC alone only released IFN-γ and overall, cytokine levels in media were lowest when MSC were cultured alone. Stimulation of MSC either by PBMC or by PL resulted in an altered expression of immunoregulatory genes. In co-culture with PBMC, the MSC gene expression of COX2, TNFAIP6, IDO1, CXCR4 and MHC2 was upregulated and VCAM1 was downregulated. In the presence of PL, COX2, TNFAIP6, VCAM1, CXCR4 and HIF1A were upregulated. Functionally, while no consistent changes were found regarding the percentage of FoxP3+ cells, MSC decreased PBMC proliferation in all media, with the strongest effect in FBS media supplemented with IL-10 or IFN-γ. This study provides further evidence that PL supports MSC functionality, including their immunoregulatory mechanisms. The results justify to investigate functional effects of MSC cultured in PL-supplemented medium on different types of immune cells in more detail.

Short-chain fatty acids: linking diet, the microbiome and immunity.

The short-chain fatty acids (SCFAs) butyrate, propionate and acetate are microbial metabolites and their availability in the gut and other organs is determined by environmental factors, such as diet and use of antibiotics, that shape the diversity and metabolism of themicrobiota. SCFAs regulate epithelial barrier function as well as mucosal and systemic immunity via evolutionary conserved processes that involve G protein-coupled receptor signalling or histone deacetylase activity. Indicatively, the anti-inflammatory role of butyrate is mediated through direct effects on the differentiation of intestinal epithelial cells, phagocytes, B cells and plasma cells, and regulatory and effector T cells. Intestinally derived SCFAs also directly and indirectly affect immunity at extra-intestinal sites, such as the liver, the lungs, the reproductive tract and the brain, and have been implicated in a range of disorders, including infections, intestinal inflammation, autoimmunity, food allergies, asthma and responses to cancer therapies. An ecological understanding of microbial communities and their interrelated metabolic states, as well as the engineering of butyrogenic bacteria may support SCFA-focused interventions for the prevention and treatment of immune-mediated diseases.

Approaches for Performance Verification Toward Standardization of Peripheral Blood Regulatory T-Cell Detection by Flow Cytometry.

Regulatory T-cell (Treg) detection in peripheral blood, based on flow cytometry, is invaluable for diagnosis and treatment of immune-mediated diseases. However, there is a lack of reliable methods to verify the performance, which is pivotal towards standardization of the Tregs assay. To conduct standardization studies and verify the performance of 3 commercially available reagent sets for the Tregs assay based on flow cytometry and agreement analysis for Treg detection across the different reagent sets. The analytical performance of Tregs assay using reagent sets supplied by 3 manufacturers was evaluated after establishing the gating strategy and determining the optimal antibody concentration. Postcollection sample stability was evaluated, as well as the repeatability, reproducibility, reportable range, linearity, and assay carryover. Agreement between the different assays was assessed via Bland-Altman plots and linear regression analysis. The relationship between the frequency of CD4+CD25+CD127low/- Tregs and CD4+CD25+Foxp3+ Tregs was evaluated. The postcollection sample stability was set at 72 hours after collection at room temperature. The accuracy, repeatability, reproducibility, and accuracy all met the requirements for clinical analysis. Excellent linearity, with R2 ≥0.9 and no assay carryover, was observed. For reportable range, a minimum of 1000 events in the CD3+CD4+ gate was required for Tregs assay. Moreover, the results for Tregs labeled by antibodies from the 3 manufacturers were in good agreement. The percentage of CD4+CD25+CD127low/- Tregs was closely correlated with CD4+CD25+Foxp3+ Tregs. This is the first study to evaluate systematically the measurement performance of Tregs in peripheral blood by flow cytometry, which provides a practical solution to verifying the performance of flow cytometry-based immune monitoring projects in clinical practice.

Nutritional interventions as modulators of the disease activity for idiopathic inflammatory myopathies: a scoping review.

Idiopathic inflammatory myopathies (IIMs) are chronic, autoimmune connective tissue diseases associated with significant morbidity and disability. Nutrients can activate the immune system and contribute to chronic low-grade inflammation (LGI). Chronic muscle inflammation leads to imbalanced pro-inflammatory and anti-inflammatory cytokines, causing inadequate nutrition, weight loss and muscle weakness during a negative cycle. Owing to its potential to modulate LGI in various diseases, the Mediterranean diet (Med Diet) has been extensively studied. This scoping review explores the nutritional implications and recommendations of the Med Diet as a treatment for immune-mediated diseases, focusing on the gaps in IIM nutritional interventions. A comprehensive literature search of the MEDLINE and EBSCO databases between September 2018 and December 2022 was performed. We identified that the Med Diet and its specific components, such as omega-3 (nω3) fatty acids, vitamin D and antioxidants, play a role in the dietary treatment of connective tissue-related autoimmune diseases. Nutritional interventions have demonstrated potential for modulating disease activity and warrant further exploration of IIMs through experimental studies. This review introduces a dietary therapeutic approach using the Med Diet and related compounds to regulate chronic inflammatory processes in IIMs. However, further clinical studies are required to evaluate the efficacy of the Med Diet in patients with IIMs. Emphasising a clinical-nutritional approach, this study encourages future research on the anti-inflammatory effects of the Med Diet on IIMs. This review highlights potential insights for managing and treating these conditions using a holistic approach.

Examining the Safety Profile of Janus Kinase (JAK) Inhibitors in the Management of Immune-Mediated Diseases: A Comprehensive Review.

Janus kinase (JAK) inhibitors have heralded a paradigm shift in the management of immune-mediated diseases. While their efficacy is well-established, the safety concerns associated with these agents, particularly regarding thromboembolic events (TE), remain a focus of extensive research and clinical scrutiny. This comprehensive literature review embarks on an exploration of the multifaceted landscape of JAK inhibitors, providing insights into their safety profiles across diverse immune-mediated diseases. The introduction highlights the transformative influence of JAK inhibitors in the treatment of immune-mediated diseases. Historically, the therapeutic arsenal for these conditions included corticosteroids, disease-modifying antirheumatic drugs (DMARDs), and biologics. The advent of JAK inhibitors has revolutionized this landscape, although concerns about their safety persist. This review strives to comprehensively evaluate their safety, amalgamating knowledge from multiple studies and trials. The subsequent sections delve into the safety of specific JAK inhibitors in the context of rheumatoid arthritis, inflammatory bowel diseases, and dermatologic conditions and their associations with venous thromboembolism. The evolving understanding of TE risk, particularly the intricate relationship between these agents and immune-mediated diseases, is meticulously unravelled. The concluding remarks underscore the dynamic nature of TE risk assessment with regard to immune-mediated diseases involving JAK inhibitors. It underscores that risk assessment is multifactorial, influenced not only by the choice of JAK inhibitor but also by the nuances of the underlying immune-mediated disease and the unique patient characteristics. This review offers a holistic perspective on TE risks associated with JAK inhibitors and contributes to the ongoing dialogue regarding their safety in the realm of immune-mediated diseases.

Dual Biologic Therapy in Patients with Rheumatoid Arthritis and Psoriatic Arthritis.

Treatment with biological agents has become standard of care in treatment of immune-mediated diseases (IMD), including rheumatoid arthritis and psoriatic arthritis. Yet, a significant proportion of patients experience loss of response to biologics, need treatment escalation, or develop side effects. During the past decade, new biologic agents with different targeted molecular pathways have been approved for treatment of IMD, introducing the possibility of concomitant dual biologic therapy. The role of dual biologic therapy targeting different inflammatory pathways has become an area of great interest in the field of IMD, addressing the unmet clinical need of patients with refractory diseases and treatment of comorbidities, such as osteoporosis, asthma, atopic dermatitis, and urticaria. Despite the increasing use of biologics as a dual therapy across different indications, there is a paucity of data concerning the safety of the simultaneous use of more than one biological agents. The purpose of this review is to summarize the current literature on the use of dual biologics in patients with rheumatoid arthritis and psoriatic arthritis, addressing the potential adverse effects associated with combination therapy, and highlighting future directions in the use of this novel therapeutic modality.

Truncated-semaphorin3A is a potential regulatory molecule to restore immune homeostasis in immune-mediated diseases.

Regulatory molecules have recently been recognized for their beneficial effects in the treatment of immune-mediated diseases, rather than using cytotoxic immune-suppressing drugs, which are associated with many unwanted side effects. Semaphorin3A (sema3A), a unique regulatory master of the immune system, was shown to be decreased in the serum of systemic lupus erythematosus (SLE) patients, in association with disease severity. Later, we were able to show its extremely beneficial effect in treating lupus nephritis in the NZB/W mice model. The mechanisms by which sema3A maintains its regulatory effect is by binding the regulatory receptor CD72 on B cells, thereby reducing the threshold of BCR signaling on B cells and reducing the production of pro-inflammatory cytokines. The aim of this study was to generate a stable sema3A molecule, easy to produce with a higher binding capacity to CD72 receptor rather than to Neuropilin-1 (NRP-1) receptor, which is expressed in many cell types. Using the crystallographic structure of parental sema3A, we synthesized a new secreted (shorter) sema3A derivative, which we called truncated sema3A (T-sema3A). The new molecule lacked the NRP-1 binding domain (the C-terminal site) and has an artificial dimerization site at position 257 (serine residue was exchanged with a cysteine residue). To facilitate the purification of this molecule we added Histidine epitope tag in frame upstream to a stop codon. This construct was transfected using a viral vector to 293HEK cells to generate cells stably expressing T-sema3A. T-sema3A is shown to be with a higher binding ability to CD72 than to NRP-1 as demonstrated by a homemade ELISA. In addition, T-sema3A was shown to be a regulatory agent which can induce the expression of IL-10 and TGF-β and reduce the secretion of pro-inflammatory cytokines such as IL-6, IFN-γ, and IL-17A from human T and B-lymphocytes. Keeping this in mind, T-sema3A is highly effective in maintaining immune homeostasis, therefore, becoming a potential agent in restoring the regulatory status of the immune system in immune-mediated diseases.

SiRNA-based nanotherapeutics as emerging modalities for immune-mediated diseases: A preliminary review.

Immune‐mediated diseases (IMDs) are chronic conditions that have an immune‐mediated etiology. Clinically, these diseases appear to be unrelated, but pathogenic pathways have been shown to connect them. While inflammation is a common occurrence in the body, it may either stimulate a favorable immune response to protect against harmful signals or cause illness by damaging cells and tissues. Nanomedicine has tremendous promise for regulating inflammation and treating IMIDs. Various nanoparticles coated with nanotherapeutics have been recently fabricated for effective targeted delivery to inflammatory tissues. RNA interference (RNAi) offers a tremendous genetic approach, particularly if traditional treatments are ineffective against IMDs. In cells, several signaling pathways can be suppressed by using RNAi, which blocks the expression of particular messenger RNAs. Using this molecular approach, the undesirable effects of anti‐inflammatory medications can be reduced. Still, there are many problems with using short‐interfering RNAs (siRNAs) to treat IMDs, including poor localization of the siRNAs in target tissues, unstable gene expression, and quick removal from the blood. Nanotherapeutics have been widely used in designing siRNA‐based carriers because of the restricted therapy options for IMIDs. In this review, we have discussed recent trends in the fabrication of siRNA nanodelivery systems, including lipid‐based siRNA nanocarriers, liposomes, and cationic lipids, stable nucleic acid‐lipid particles, polymeric‐based siRNA nanocarriers, polyethylenimine (PEI)‐based nanosystems, chitosan‐based nanoformulations, inorganic material‐based siRNA nanocarriers, and hybrid‐based delivery systems. We have also introduced novel siRNA‐based nanocarriers to control IMIDs, such as pulmonary inflammation, psoriasis, inflammatory bowel disease, ulcerative colitis, rheumatoid arthritis, etc. This study will pave the way for new avenues of research into the diagnosis and treatment of IMDs.

P631 Impact of Covid-19 pandemic on initiation of immunosuppressive treatment in immune-mediated diseases. -A nationwide, retrospective study

BackgroundImmunosuppressive and biological medications are a mainstay in the treatment of immune-mediated diseases, such as inflammatory bowel diseases (IBD), rheumatic diseases, and psoriasis. However, the COVID-19 pandemic caused concerns over the safety of these drugs pertaining to risk and severity of infection with SARS-CoV2. Moreover, pandemic mitigation strategies may negatively impacted treatment start with immunosuppressive and biological treatment fostering worse long-term disease outcomes. The aim of this study was to examine the impact of the COVID-19 pandemic on new starts of immunosuppressive and biological treatment in Austria.MethodsWe conducted a retrospective analysis with a 4-year observation period from 2017 to 2020 on real-world data on prescriptions for immune-mediated diseases of the Austrian health insurance funds covering 98% of the Austrian population. Data from all patients with incident biologic or conventional immunosuppressive treatment (Table 1) were included. Incidence of biologic (including small molecules) and immunosuppressive therapy was defined as all first prescriptions of one of the listed substances from 2017. The incidence rate for biologic and immunosuppressive treatments was recorded monthly in 2020 and compared with the three previous years (2017 – 2019). ResultsDuring the first lockdown in Austria in spring 2020 (week 12 – week 20), there was a significant decrease in the overall starts of biologic (including small molecules) and immunosuppressive treatments (both p<0.0001), especially in April (Figure 1 and 2). After that lockdown, new starts of immunosuppressive and biological treatments rapidly re-achieved pre-lockdown levels despite higher infection rates with SARS-CoV-2 and subsequent lockdown periods (Figure 3). Independent from the COVID-19 pandemic, we observed a continuous increase of biological medication (bDMARDs) and small molecules (p<0.0001) and a decrease of conventional immunosuppressive medications (cDMARDS) (p<0.0120) during all observed years (Figure 1 and 2) ConclusionIn patients with immune-mediated diseases in Austria the COVID-19 pandemic led to a significant decrease of newly started immunosuppressive and biological treatments only during the first lock-down. Over the last four years, we can observe a continuous increase of small molecules and biological medication as well as a continuous decrease of conventional immunosuppressive medication.

Na-AIP-1 secreted by human hookworms suppresses collagen-induced arthritis.

Proteins from helminths have been posed as new immunomodulatory agents with exciting potential in the treatment of immune-mediated diseases including rheumatoid arthritis (RA). In this study we assess the effects of a helminthic excretory/secretory (ES) protein Na-AIP-1 as monotherapy and in combination with methotrexate (MTX) in the well-described collagen-induced arthritis (CIA) model of RA. CIA was induced in DBA/1J mice which were treated after the onset of arthritis with Na-AIP-1 monotherapy, MTX or Na-AIP-1 + MTX. The clinical scores for weight, arthritis and paw width were recorded along with joint histology as outcome measures. For the clinical parameters of weight, paw score and paw width, none of the Na-AIP-1 monotherapy, MTX therapy or Na-AIP-1 + MTX combination therapy groups displayed any significant difference when compared to the arthritis control. However, a significant reduction in histological score was identified after both monotherapy (Na-AIP-1: 0.83 ± 0.24 vs Arthritis control: 5.58 ± 1.49, p = 0.0277) and combination therapy (Na-AIP-1 + MTX: 0.55 ± 0.28 vs Arthritis control: 5.58 ± 1.49, p = 0.0233) when compared to arthritis control. Furthermore, Na-AIP-1 as both monotherapy (Na-AIP-1: 0.83 ± 0.24 vs MTX: 5.73 ± 1.82 p = 0.0261) and combination therapy (Na-AIP-1 + MTX: 0.55 ± 0.28 vs MTX: 5.73 ± 1.82, p = 0.0221) also significantly reduced histological score when compared to MTX monotherapy. Na-AIP-1 significantly reduced joint pathology in CIA. The hookworm protein Na-AIP-1 seems to be effective in the treatment of RA as monotherapy and when dosed together with MTX, constituting a potential new candidate for drug development. Research should focus on elucidating the mechanism of Na-AIP-1 action as a means to identify novel targets for therapeutics and to further our current understanding of immunobiology in RA.

Bruton's Tyrosine Kinase (BTK) Inhibitors and Autoimmune Diseases: Making Sense of BTK Inhibitor Specificity Profiles and Recent Clinical Trial Successes and Failures.

Clinical development of BTK kinase inhibitors for treating autoimmune diseases has lagged behind development of these drugs for treating cancers, due in part from concerns over the lack of selectivity and associated toxicity profiles of first generation drug candidates when used in the long term treatment of immune mediated diseases. Second generation BTK inhibitors have made great strides in limiting off-target activities for distantly related kinases, though they have had variable success at limiting cross-reactivity within the more closely related TEC family of kinases. We investigated the BTK specificity and toxicity profiles, drug properties, disease associated signaling pathways, clinical indications, and trial successes and failures for the 13 BTK inhibitor drug candidates tested in phase 2 or higher clinical trials representing 7 autoimmune and 2 inflammatory immune-mediated diseases. We focused on rheumatoid arthritis (RA), multiple sclerosis (MS), and systemic lupus erythematosus (SLE) where the majority of BTK nonclinical and clinical studies have been reported, with additional information for pemphigus vulgaris (PV), Sjogren’s disease (SJ), chronic spontaneous urticaria (CSU), graft versus host disease (GVHD), and asthma included where available. While improved BTK selectivity versus kinases outside the TEC family improved clinical toxicity profiles, less profile distinction was evident within the TEC family. Analysis of genetic associations of RA, MS, and SLE biomarkers with TEC family members revealed that BTK and TEC family members may not be drivers of disease. They are, however, mediators of signaling pathways associated with the pathophysiology of autoimmune diseases. BTK in particular may be associated with B cell and myeloid differentiation as well as autoantibody development implicated in immune mediated diseases. Successes in the clinic for treating RA, MS, PV, ITP, and GVHD, but not for SLE and SJ support the concept that BTK plays an important role in mediating pathogenic processes amenable to therapeutic intervention, depending on the disease. Based on the data collected in this study, we propose that current compound characteristics of BTK inhibitor drug candidates for the treatment of autoimmune diseases have achieved the selectivity, safety, and coverage requirements necessary to deliver therapeutic benefit.

OP0123 SAFETY PROFILE OF JAK-INHIBITORS VERSUS TNF-INHIBITORS IN REAL-WORLD CLINICAL PRACTICE: DATA FROM A MULTICENTER REGISTER

Background:JAK-inhibitors (JAKi) are small molecules emerging as a promising treatment for immune mediated diseases. Data from clinical trials are very promising. But long-term observational studies, with patients with diverse clinical backgrounds are required to confirm safety profile.Objectives:To compare the safety profile of JAKi versus TNF antagonists (anti-TNF) in a multicenter real world dataset.Methods:Data of patients enrolled in BIOBADASER 3.0 up to November 2020 with the start of anti-TNF or JAKi were analysed. Adverse events (AE) were classified according to Meddra dictionary (v19.0). For each group, demographic, clinical variables and ncidence rate ratios of AE per 1000 patients-year (PYs) and 95% confidence interval were estimated.Results:A total of 3,729 patients on anti-TNF (5,306) or JAKi (493) were analyzed. Patients on JAKi were older and with a mean disease duration of 10 years. JAKi were prescribed as first line treatment only in 24% of patients. The main reason of stopping treatment was ineffectiveness (53-59%) followed by adverse events (25-34%) in both groups. Survival during first year was similar between groups.Table 1.Patient characteristics and adverse events by treatment groupAnti-TNFJAKiMean age start of treatment (SD), yrs50.8 (12.6)57.6 (11.9)Female, n (%)3122 (58.8)392 (79.5)Disease duration, median (IQR)7.0 (2.7-13.7)9.9 (4.9-16.8)First line biologic, n (%)2614 (49.3)117 (23.7)Rheumatoid Arthritis1385 (41.1)339 (95.2)Ankylsosing Spondylitis1031 (30.6)1 (0.3)Psoriatic Arthritis957 (28.4)16 (4.5)DAS28-ESR4.3 (1.4)4.7 (1.4)Survival first year (IC 95%)73.3 (71.9-74.6)69.7 (66.0-73.0)Charlson Index, mean (SD)1.9 (1.3)2.3 (1.6)Reason to stop therapy (n)*: Lack of efficacy1534 (53.2)57 (58.8) Adverse event723 (25.1)33 (34.0)Adverse events (AE)*Serious infections14.2 (12.4-16.2)33.2 (19.3-57.3)Herpes zoster5.7 (4.6-7.1)12.8 (5.3-30.7)Tuberculosis0.7 (0.4-1.3)0.0 (0.0-0.0)Malignancy/Neoplasia10.2 (8.7-11.9)15.3 (6.9-34.2)Cardiac events13.9 (12.2-16.0)30.7 (17.4-54.0)GI perforation1.2 (0.8-1.9)10.2 (3.8-27.3)Vascular events9.8 (8.3-11.5)25.6 (13.8-47.5)*Data show the incidence rate ratio per 1000 patient-years (PYs; 95% CI)Conclusion:Serious infections and herpes zoster tend to be more frequent in patients on JAKi. However patients on JAKi were older, presented higher comorbidity and have a longer disease duration.Disclosure of Interests:None declared

Antitumor Activity and Immunomodulatory Effects of Ficus carica Latex

Introduction: There are limited studies on the anti-cancer and immunomodulatory effects of the fig fruit latex. In this study, we aimed to investigate the effect of fig fruit latex on several cancer cell lines as well as its effect on lymphocytes proliferation and cytokines secretion.&#x0D; Methods: After preparing a methanolic extract from fig latex, its effect on various cancer cell lines including Fen (bladder cancer), K562 (myeloid leukemia), Hela (cervix carcinoma), Jurkat (lymphoid leukemia) and Raji (lymphoma) was examined by MTT colorimetric assay. For evaluating the effects of extract on lymphocyte proliferation and viability, BrdU assay and flow cytometry staining were used. Cytokine secretion was measured by ELISA assay.&#x0D; Results: The extract showed the strongest activity against K562 cell line (IC50, 234 µg/ml) and the least activity against Hela cells (IC50 &gt;1000 µg/ml). On evaluation of the immunomodulatory effect of fig by BrdU assay, a reduction in lymphocytes proliferation by increasing the concentration of the extract was observed; proliferation index from 1.2±0.06 at 0.1 µg/ml of the extract reached to 0.13±0.2 at 800 µg/ml. In flow cytometry analysis, a significant cytotoxic effect at concentrations ≥400 µg/ml was observed. The extract at 100 and 200µg/ml had the ability to reduce secretion of interferon (IFN)-γ and interleukin (IL)-4 cytokines.&#x0D; Conclusion: Fig latex extract showed cytotoxicity on different cells particularly K562 leukemia cells which implied its anticancer activity. This extract at lower concentrations reduced lymphocytes proliferation and cytokine production which showed its immunoinhibitory effects and suggested its possible beneficial in treatment of immune-mediated diseases.

Latest advances to enhance the therapeutic potential of mesenchymal stromal cells for the treatment of immune-mediated diseases.

Mesenchymal stromal cells (MSCs) present the capacity to secrete multiple immunomodulatory factors in response to their microenvironment. This property grants them a golden status among the novel alternatives to treat multiple diseases in which there is an unneeded or exaggerated immune response. However, important challenges still make difficult the clinical implementation of MSC-based therapies, being one of the most remarkable the lack of efficacy due to their transient immunomodulatory effects. To overcome this issue and boost the regulatory potential of MSCs, multiple strategies are currently being explored. Some of them consist of ex vivo pre-conditioning MSCs prior to their administration, including exposure to pro-inflammatory cytokines or to low oxygen concentrations. However, currently, alternative strategies that do not require such ex vivo manipulation are gaining special attention. Among them, the recreation of a three dimensional (3D) environment is remarkable. This approach has been reported to not only boost the immunomodulatory potential of MSCs but also increase their in vivo persistence and viability. The present work revises the therapeutic potential of MSCs, highlighting their immunomodulatory activity as a potential treatment for diseases caused by an exacerbated or unnecessary immune response. Moreover, it offers an updated vision of the most widely employed pre-conditioning strategies and 3D systems intended to enhance MSC-mediated immunomodulation, to conclude discussing the major challenges still to overcome in the field.

Pharmacokinetics and Pharmacodynamics of Immediate- and Modified-Release Mycophenolic Acid Preparations in Healthy Beagle Dogs

Background: Mycophenolic acid (MPA) is a broad-acting immunomodulating agent that may be therapeutically beneficial for the treatment of immune-mediated diseases in canine patients.Objectives: To determine the suppressive effects of MPA on T-cell proliferation, and to assess the feasibility of a canine-specific q24 h modified-release MPA formulation (OKV-1001b).Animals: Fifteen healthy purpose-bred male beagle dogs.Methods: Two nearly identical open-label fifteen-day studies were conducted in which dogs were randomized to receive mycophenolate mofetil (MMF; 10 mg/kg q12h), or two doses of OKV-1001b (270 mg and 180 mg; q24h). Serial pharmacokinetic (PK) and pharmacodynamic (PD) samples were collected on Days 1, 8, and 15. MPA plasma concentrations were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS), while an ex vivo T-cell proliferation assay assessed PD effects. Dogs were continuously monitored for evidence of side effects and gastrointestinal tolerability.Results: MPA induced inhibition of T-cell proliferation was observed following administration of all MPA preparations in a clear concentration-dependent manner. The PK/PD relationship was maintained across all days and time-points. Data generated herein suggest that MPA plasma concentrations above 600 ng/mL achieve at least 50% inhibition of T-cell proliferation.Conclusions and Clinical Importance: MPA holds therapeutic potential for treating dogs with immune-mediated disease, but clinical trials will be necessary to determine its safety and efficacy in naturally occurring disease. Likewise, q24h oral modified release MPA preparations that maintain MPA plasma concentrations between 600 and 1,000 ng/mL are warranted for further studies in client-owned dogs.

Engineered Type-1 Regulatory T Cells as Cellular Therapy for Treatment of Immune Mediated Diseases

Abstract Type 1 regulatory cells (Tr1) are a promising cellular product for suppression of effector T cells in immune mediated diseases, including graft-versus-host-disease (GvHD) in allogeneic hematopoietic stem cell transplantation (allo-HSCT) (Roncarolo et al. Immunity 2018). We have developed an in vitro protocol to produce Tr1 cells by lentiviral transduction of the human IL10 with a constitutive promoter into human CD4+ T cells (Locafaro et al. Molecular Therapy 2017). These engineered Tr1 cells, called LV-10, acquire a characteristic Tr1 cytokine profile (IL-10 and IFN-g high, IL-4 low and expression of intracellular perforin and granzyme B). In vitro, LV-10 cells suppress the proliferation of responder CD4+ T cells upon activation by allogeneic dendritic cells. LV-10 cells also degranulate in response to and kill myeloid cells, including myeloid blasts from patients with acute myeloid leukemia, through a granzyme B- and perforin-dependent mechanism. Interestingly, the ability to degranulate and kill myeloid cells is not present when LV-10 are activated and expanded with anti-CD3 and anti-CD28 coated beads, suggesting that signals beyond TCR, CD28, and IL-10 receptor pathway activation are necessary to reprogram LV-10 cells into cytotoxic cells. In vivo, LV-10 cells injected into NSG mice do not induce xeno-GvHD, in contrast to control CD4+ T cells. In addition, LV-10 cells suppress CD4-induced xeno-GvHD and prevent expansion of myeloid leukemic cells. Experiments are ongoing to compare the potency and in vivo survival of allogeneic vs autologous LV-10 cells. These findings demonstrate the promise of using LV-10 to treat immune mediated diseases, including GvHD in AML patients receiving allo-HSCT.

JAK Inhibitors Safety in Ulcerative Colitis: Practical Implications.

Janus kinase inhibitors [JAKi] are a new class of small molecule drugs that modulate inflammatory pathways by blocking one or more JAK receptors, and are increasingly being used in the treatment of immune-mediated diseases. Tofacitinib, a non-selective JAKi, is now approved for moderate-to-severe ulcerative colitis [UC] that is refractory or intolerant to tumour necrosis factor inhibitors [TNFi]. Whereas tofacitinib is associated with the advantages of oral administration, rapid onset of action, and lack of immunogenicity over TNFi, there are many safety considerations to take into account such as the risk of thromboembolism, infections, and hyperlipidaemia: each with specific nuances pertaining to prevention and monitoring strategies. Considerations such as pregnancy, breastfeeding, and history of malignancy also are to be navigated with utmost caution, given that very few data are available for guidance. With the use of JAKi in the real world progressively over time, safety implications will become more lucid, including caveats pertaining to JAK selectivity and gut-selective JAKi, as well as mechanistic data pertaining to adverse effects. This Viewpoint serves as a practical guide for clinicians managing inflammatory bowel disease [IBD] patients to navigate safety concerns around JAKi, including preventive and monitoring strategies.

Pyridoxal-5'-Phosphate Promotes Immunomodulatory Function of Adipose-Derived Mesenchymal Stem Cells through Indoleamine 2,3-Dioxygenase-1 and TLR4/NF-κB Pathway.

Adipose-derived mesenchymal stem cells (A-MSCs) are promising cellular therapies for the treatment of immune-mediated diseases. Non-gene editing technologies can improve the immune regulatory function of A-MSCs. Our preliminary experiments revealed that an active form of vitamin B6—pyridoxal-5′-phosphate (PLP)—plays an important role in regulating gene expression and cytokine secretion in A-MSCs in vivo. To further clarify the effect of PLP on receptors and cytokines related to the immune regulatory function of A-MSCs, a series of experiments were designed to verify the relationships between PLP and A-MSCs in vitro. Initially, A-MSCs were obtained, and cytokine secretion and the expression of IDO1, NF-κB, and Toll-like receptors in PLP-stimulated A-MSCs were evaluated. In addition, coculture was used to detect A-MSCs-mediated apoptosis of CD3+CD8+ T lymphocytes. These results showed that A-MSCs stimulated with PLP were highly proliferative, consistent with their pluripotent capacity. Further, the surface receptors TLR3, TLR4, IDO1, and NF-κB were upregulated, while TLR6 was downregulated. Concurrently, A-MSCs preconditioned with PLP had the greatest inhibitory effect on CD3+CD8+ T lymphocyte proliferation, indicating that PLP altered the immune regulatory function of A-MSCs through the regulation of TLRs and IDO1 expression.

Infliximab–Tumor Necrosis Factor Complexes Elicit Formation of Anti-Drug Antibodies

Some patients develop anti-drug antibodies (ADAs), which reduce the efficacy of infliximab, a monoclonal antibody against tumor necrosis factor (TNF), in the treatment of immune-mediated diseases, including inflammatory bowel diseases. ADAs arise inconsistently, and it is not clear what factors determine their formation. We investigated features of the immune system, the infliximab antibody, and its complex with TNF that might contribute to ADA generation. C57BL/6 mice were given injections of infliximab and recombinant human TNF or infliximab F(ab')2 fragments. Blood samples were collected every 2-3 days for 2 weeks and weekly thereafter for up to 6 weeks; infliximab-TNF complexes and ADAs were measured by enzyme-linked immunosorbent assay (ELISA). Intestinal biopsy and blood samples were obtained from patients having endoscopy who had received infliximab therapy for inflammatory bowel diseases; infliximab-TNF complexes were measured with ELISA. Infliximab-specific plasma cells were detected in patient tissue samples by using mass cytometry. We studied activation of innate immune cells in peripheral blood mononuclear cells (PBMCs) from healthy donors incubated with infliximab or infliximab-TNF complexes; toll-like receptors (TLRs) were blocked with antibodies, endocytosis was blocked with the inhibitor PitStop2, and cytokine expression was measured by real-time polymerase chain reaction and ELISAs. Uptake of infliximab and infliximab-TNF complexes by THP-1 cells was measured with confocal microscopy. Mice given increasing doses of infliximab produced increasing levels of ADAs. Blood samples from mice given injections of human TNF and infliximab contained infliximab-TNF complexes; complex formation was associated with ADA formation with an area under the curve of 0.944 (95% confidence interval, 0.851-1.000; P= .003). Intestinal tissues from patients, but not blood samples, contained infliximab-TNF complexes and infliximab-specific plasma cells. Incubation of PBMCs with infliximab-TNF complexes resulted in a 4.74-fold increase in level of interleukin (IL) 1β (IL1B) messenger RNA (P for comparison= .005), increased IL1B protein secretion, and a 2.69-fold increase in the expression of TNF messenger RNA (P for comparison= 0.013) compared with control PBMCs. Infliximab reduced only IL1B and TNF expression. Antibodies against TLR2 or TLR4 did not block the increases in IL1B or TNF expression, but endocytosis was required. THP-1 cells endocytosed higher levels of infliximab-TNF complexes than infliximab alone. In mice, we found ADA formation to increase with dose of infliximab given and concentration of infliximab-TNF complexes detected in blood. Based on studies of human intestinal tissues and blood samples, we propose that infliximab-TNF complexes formed in the intestine are endocytosed by and activate innate immune cells, which increase expression of IL1B and TNF and production of antibodies against the drug complex. It is therefore important to optimize the infliximab dose to a level that is effective but does not activate an innate immune response against the drug-TNF complex.

Serum Vitamin D Metabolites and CXCL10 Concentrations Associate With Survival in Dogs With Immune Mediated Disease.

Background: Low vitamin D increases the risk of immune-mediated disease (IMD) in human beings and rodent models. Vitamin D metabolites, particularly 1,25(OH)2D3, modulate gene expression of immune cells and may attenuate immune pathways that drive IMD.Hypothesis/objectives: Our primary hypothesis was that serum 25(OH)D3 and 1,25(OH)2D3, are reduced in patients with IMD and associate with poorer outcomes. We secondarily hypothesized serum 24,25(OH)2D3 would not be associated with disease or outcome. We also measured serum CXCL10 concentrations to determine if an increase occurs in dogs with IMD and in association with poorer survival.Animals: We enrolled dogs diagnosed with IMD (n = 29) and healthy control dogs (n = 9) in the study with informed client consent.Methods: Serum was collected and stored at −80°C until analyses. Serum vitamin D metabolites were measured by LC-MS/MS by an accredited laboratory. A commercially available canine-specific ELISA kit measured serum CXCL10.Results: Serum 25(OH)D3 and 1,25(OH)2D3 were significantly reduced in dogs (n = 25) with IMD. Serum CXCL10 concentrations undetectable in all controls, and were 30 times higher overall in IMD dogs (n = 25; P = 0.004). CXL10 was, however, undetectable in 40% of dogs with IMD. Of the 60% of IMD dogs with increased CXCL10 concentrations, 5/25 had concentrations at the upper limit of quantification. The survival of those five dogs was significantly (P = 0.049) shorter (72 days) than all other dogs with IMD with measured CXCL10 concentrations. The median survival time (MST) for dogs with 25(OH)D3 concentrations ≤ the median was 106 days, while dogs with concentrations of 25(OH)D3 > the median did not achieve an MST.Conclusions and clinical importance: Serum 25(OH)D3 and 1,25(OH)2D3, but not 24,25(OH)2D3 levels are reduced dogs with IMD. Vitamin D metabolites and CXCL10 may be useful prognostic markers and may be targets for adjunct therapy in canine IMD. These data support the future investigation of vitamin D analogs in the treatment of canine IMD.